Maderspach K, Fajszi C
Biochim Biophys Acta. 1982 Nov 22;692(3):469-78. doi: 10.1016/0005-2736(82)90399-6.
Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.
通过[3H]二氢-L-阿普洛尔（[3H]DHA）的特异性结合，对原代培养的鸡、大鼠和小鼠胚胎脑完整细胞中的β-肾上腺素能受体进行了研究。将结果与来自细胞培养物或用于制备培养物的前脑组织的破碎细胞制剂的受体结合情况进行了比较。对[3H]DHA与活鸡脑细胞结合的详细分析揭示了一个高亲和力、立体选择性的β-肾上腺素能型结合位点。平衡测量表明结合反应具有明显的正协同性。通过将希尔方程直接拟合到测量数据，得到Bmax = 12.01 fmol/10(6)细胞（7200个位点/细胞）、Kd = 60.23 pM以及希尔系数n = 2.78的值。与L-阿普洛尔竞争的动力学证实了结合的明显协同特性，在低配体浓度下产生最大曲线。根据结合结果，结合反应的速率常数估计为k+ = 8.31×10(7) M-1×min-1和k- = 0.28 min-1，根据解离数据，k- = 0.24 min-1。结合动力学支持结合的协同性，得到希尔系数n = 1.76；Kd，作为(k-/k+)1/n被发现为101 pM。对[3H]DHA与大鼠和小鼠活脑细胞的平衡结合分析分别得到Bmax = 13.04 fmol/10(6)细胞（7800个位点/细胞）、Kd = 43.85 pM和n = 2.52，以及Bmax = 8.08 fmol/10(6)细胞（4800个位点/细胞）、Kd = 46.70 pM和n = 1.63，也证实了哺乳动物对象中β受体的明显协同性。发现[3H]DHA与鸡、大鼠或小鼠脑培养物或前脑组织的破碎细胞制剂的平衡结合是非协同性的，希尔系数n = 1，Kd在1 - 2 nM范围内，Bmax为10(3) - 10(4)个位点/细胞。我们的研究结果表明，细胞破碎会导致β受体结合动力学和结合位点亲和力发生显著变化，尽管受体数量保持不变。
